Abstract

The purpose of this study was to investigate the clinical usefulness of
the color Doppler vascularity index (CDVI) in patients with colon
cancer before surgery. Forty-four patients with sonographically visible
tumor mass of colon cancer were investigated. The CDVI of each tumor
was determined using transabdominal color Doppler ultrasound. The CDVI
was defined as the ratio of the number of the colored pixels within a
tumor section to the number of total pixels in that specific tumor
section and was calculated by using Encomate software (Electronic
Business Machine Co. Ltd., Taipei, Taiwan). The correlation between the
CDVI and clinicopathological factors, mode of recurrence, and patient
survival was studied. For comparison, microvessel density (the mean
number of microvessels in three areas of highest vascular density at×
200 magnification) of the tumors of these 44 patients was also
evaluated by using immunohistochemical staining of surgical specimens
with anti-CD34. The microvessel density was not correlated with Dukes’
classification, clinicopathological factors, and survival. The CDVI was
significantly higher in the patients with lymph node metastases and
vascular invasion than in those without such metastases and invasion
(P = 0.006 and P = 0.0098, respectively). Moreover, in patients with a high CDVI
(>15%) and positive vascular invasion, survival was significantly
poorer than in those with low CDVI (≤15%) and negative invasion
(P = 0.0037 and 0.0039, respectively).
Multivariate analysis indicated that liver metastasis, vascular
invasion, and CDVI are independent prognostic factors in the patients
with colon cancer. According to the mode of recurrence in 36 patients
who underwent curative resection, the frequency of the distant organ
recurrence was significantly higher in the high CDVI group (40%) than
in the low CDVI group (0%). The CDVI is a good preoperative indicator
of recurrence and patient survival in colon cancer. Thus, the CDVI may
be helpful in stratifying patients for adjuvant therapy.

INTRODUCTION

Angiogenesis is essential for the growth of solid tumors measuring
more than a few millimeters
(1)
. It permits rapid tumor
growth and potential presence of tumor metastasis
(2, 3)
.
Several studies have reported an association between the degree of
angiogenesis and the clinicopathological factors and prognosis of
patients with various solid tumors, such as breast
(4,
5,
6)
,
lung
(7)
, prostate
(8)
, head and neck
(9)
, and gastrointestinal cancer
(10, 11)
.
All these studies were accomplished on tissue sections retrospectively
obtained from surgical specimens. However, an “in vivo “
method to assess tumor angiogenesis is highly desirable for diagnostic
purpose, treatment planning, and follow-up.

With the current technique of color Doppler sonography, tumor
vascularity can be assessed in vivo(12)
. The
correlation of the color Doppler vascular signals with the angiographic
and histological findings has also been shown in tumors of various
human organs
(13, 14)
. Incremental angiogenesis could be
demonstrated in the tumorigenesis of ovarian and endometrial
malignancies with color Doppler ultrasound
(15, 16)
.
Vascularity index is a new ultrasound parameter for evaluating in
vivo angiogenesis. Several reports have revealed that the
vascularity index could be used to differentiate the nature of neck
lymph node
(17)
and had a good correlation with status of
lymph node metastasis in cervical carcinoma
(18)
.

Conventional transabdominal sonography has become increasingly
important not only in evaluating diseases of solid organ but also in
diagnosis of the gastrointestinal diseases, such as colon cancer
(19,
20,
21,
22)
. Therefore, tumoral vascularity may also be
assessed by transabdominal color Doppler ultrasound. Combining
transabdominal color Doppler ultrasound and the concept of the
vascularity index, we defined a new parameter for tumor angiogenesis,
CDVI
3
.

To investigate the clinical usefulness of the CDVI in colon cancer,
this study was conducted to evaluate the correlation between the CDVI
and clinicopathological factors, recurrence, and survival in patients
with sonographically visible tumor. For comparison, MVD, determined
immunohistochemically, was also evaluated.

PATIENTS AND METHODS

Patients.

A total of 56 patients with colon cancer, who had undergone colectomy
at our institution from January 1996 to February 1999, were included in
this study. They were all proved to have adenocarcinomas by
colonofibroscopic biopsies. Abdominal ultrasound was performed before
operation to evaluate liver and other i.p. metastases routinely without
hydrocolonic preparation after overnight fasting. The tumor mass of
colon cancer could be delineated by trans-abdominal
ultrasound in 44 patients (79%) who constituted the population of this
study. These 44 patients ranged in age from 41–82 yr (average age,
64.4 yr); there were 30 men and 14 women (Table 1
⇓
). No patient had received chemotherapy and radiotherapy before surgery.
Tumors were divided into two histological subgroups: differentiated and
undifferentiated types. Depth of invasion (within the wall or through
the wall), lymph node metastasis (negative or positive), and liver
metastasis (negative or positive) were also evaluated. They were staged
according to Dukes’ classification. Thirty-six patients received
curative surgery, whereas the remaining eight patients underwent
palliative resection only. All patients staged Dukes’ B2 or later
received postoperative chemotherapy with the same regimen of
5-fluorouracil and leucovorin. The patients were followed up from 6–34
months after surgery. The follow-up intervals were calculated as
survival intervals after surgery. Different modes of metastasis were
confirmed by diagnostic imaging and surgery.

The scanner we used was a color Doppler ultrasound unit (HDI 3000;
Advanced Technology Laboratories, Bothell, WA) with a 2–5-MHz curved
array and a 5–10-MHz broad band linear array transducers. Settings of
the color Doppler ultrasound were standardized for the highest
sensitivity in the absence of apparent noise by using a medium wall
filter, pulsed repetition frequency of 1000 Hz, moderate-to-long
persistence, and a slow sweep technique. Under these conditions, the
lowest possible measurable velocity was claimed below 5 cm/s. Focusing
depth was set between 1.5 and 4 cm. The 2–5-MHz curved array
transducer was used to search the site of colon cancer after routine
abdominal sonographic examination, then changed the transducer to the
5–10-MHz linear one for color Doppler evaluation of the tumor. Each
tumor was scanned thoroughly, and tangential scanning was made to avoid
the intraluminal air interference. The color window was set to cover
the whole tumor on the screen. The tumor of the colon cancer was then
scanned carefully in all directions, and the tumor section with
subjectively maximal color signals was captured and stored for later
analysis. Each tumor was scanned three times, thus three tumor sections
with maximal color signals were available for quantitative analysis.
After the examination, the previously stored images were retrieved and
displayed on the monitor. The tumor margin was contoured using a cursor
(Fig. 1
⇓
). Quantification of the vascular color signals within the demarcated
tumor area was then automatically performed by a special software
(Encomate; Electronic Business Machine Co., Ltd., Taipei, Taiwan). The
results were expressed as the “CDVI” (the number of colored pixels
within the tumor section/the number of total pixels in that particular
tumor section). For each tumor, the mean of the CDVI of three
representative tumor sections was used for statistical analysis.

CDVI assessment within the colon cancer. A,
the color window was set to cover the whole tumor on the screen and
stored for later quantitative analysis. B, the tumor
margin was contoured using a cursor. Quantification of the vascular
color signals within the demarcated tumor was then automatically
executed by special software called Encomate (Electronic Business
Machine Co., Ltd., Taipei, Taiwan). C, the results were
expressed as the “CDVI” (the number of colored pixels within the
tumor section/the number of total pixels in that particular tumor
section).

Microvessel Staining and Evaluation.

The paraffinized tumor blocks of 44 patients whose colon cancers could
be visualized by ultrasound were stained for endothelial cell CD34
antigen using the labeled streptavidin-biotin after antigen retrieval
(Fig. 2
⇓
). Briefly, deparaffinized sections were heated in a pressure cooker.
After endogenous peroxidase was blocked with 3% hydrogen peroxide in
the section, each section was incubated with nonimmune horse serum. The
sections were incubated in anti-CD34 monoclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) at a dilution of 1:20 or the
control nonimmune serum at 4°C overnight. The sections were incubated
with link antibodies, followed by peroxidase-conjugated streptavidin
complex (LSAB kit; DAKO Corporation, Carpinteria, CA). The peroxidase
activity was visualized with diaminobenzidine tetrahydroxychioride
solution (DAKO Corporation) as the substrate. The sections were lightly
counterstained with hematoxylin. After screening the areas with intense
neovascularized spots at low power field (×100), microvessels in the
area with the highest number of discrete microvessels were counted in a×
200 field. Three separate intense neovascularized areas were
assessed, and the mean was calculated as the MVD of each tumor
evaluated.

Immunohistochemical staining for CD34 in colon cancer
tissues (original magnification, ×200). Microvessels are represented
by brown clusters, which stand out sharply from other
tissues.

Statistics.

The relationship between MVD, CDVI, and the various clinicopathological
factors was examined by χ2 test. One-way ANOVA
was used to test the correlation among different Dukes’ stages.
Survival curves were calculated using the Kaplan-Meier method and
analyzed by the log-rank test. The CDVI and clinicopathological
variables influencing survival were assessed by the Cox proportional
hazards model. The mode of recurrence was examined by Fisher’s exact
test. Statistical significance was defined as P < 0.05.

RESULTS

The CDVI of the 44 sonographically visible colon cancers ranged
from 2.0–34%, with a mean value of 15.4%, whereas the MVD ranged
from 38.7–150.0, with a mean value of 81.5. Table 2
⇓
shows the correlation between the CDVI and the MVD, with various
clinicopathological factors. There was no statistically significant
association between the MVD and clinicopathological factors as tested
in this study. However, the CDVI in the patients with lymph node
metastases and vascular invasion was significantly higher than in those
without lymph node metastases (P = 0.006) and
vascular invasion (P = 0.0098). The CDVI of
undifferentiated tumors was also significantly higher than that of
differentiated tumors (P = 0.0022).

The prognosis of the 44 patients was then analyzed. Because the mean
MVD of these patients was 81.5, we, therefore, classified them into two
subgroups: one group of MVD >82 and one group of MVD ≤82. The mean
CDVI of these patients was 15.4%. Accordingly, the patients were
divided into two subgroups: one group of high CDVI (>15%) and one
group of low CDVI (≤15%). The survival rates were calculated using
the Kaplan-Meier method. The survival of the group with high MVD and
that with low MVD was not significantly different (Fig. 4
⇓
). On the contrary, the survival of the group with high CDVI was
significantly (P = 0.0037) worse than that
with low CDVI (Fig. 5
⇓
). The effects of variables presumably associated with patient survival
were studied by multivariate analysis using a Cox model. As a result,
liver metastasis, vascular invasion, and vascularity index were
independent prognostic factors (Table 3
⇓
).

Among the 36 patients who underwent curative resection, 10 experienced
disease recurrence. The relationship between CDVI and recurrence is
shown in Table 4
⇓
. The recurrence rate in the high CDVI group was 45%, which was
significantly higher than the rate in the low CDVI group (6.3%). As to
the mode of recurrence, the frequency of distant organ recurrence such
as liver, lung, and bone marrow after curative resection was
significantly higher in the high CDVI group (40%) than in the low CDVI
group (0%; P = 0.005).

DISCUSSION

In this study, the CDVI was significantly associated with tumor
differentiation, lymph node metastasis, and tumor vascular invasion and
was also an independent prognostic factor. Furthermore, according to
the mode of recurrence, distant organ recurrence was significantly more
frequent in the high CDVI group. However, the MVD of colon cancer was
not correlated with tumor differentiation, tumor invasion depth, lymph
node metastasis, liver metastasis, tumor vascular invasion, and patient
survival.

Current ultrasound technology is not capable of detecting tumor
neovascularization itself (approximately 15 μm or less in diameter),
which was usually demonstrated immunohistochemically
(23)
. The color Doppler signals seen within tumor
represented the larger vessels (approximately 100 μm or more in
diameter), possibly intratumoral arterioles, venules, and
arteriole-vanule shunting
(23, 24)
. We hypothesized that
the more neovascularization exists, the more supplying intratumoral
arterioles and draining venules will be present. Thus, the CDVI, by
quantitatively depicting the larger supplying arterioles and draining
venules, can reflect the extent of global neovascularization of a
tumor.

Liotta et al.(25, 26)
developed a tumor
perfusion study with C57BL/6J male mice to determine the dynamics of
tumor growth, density, and size distribution of perfused tumor vessels,
entry rate of tumor cells and tumor cell clumps, and number of
pulmonary metastases. They found that the tumor vessel size and density
are important determinants of the size of tumor cell clumps and
concentration of effluent tumor cells released into the circulation.
The number of intratumoral vessels of diameter ≥100 μm was important
for passage of tumor cell clumps, which produced a significantly
greater number of metastases than did the same number of cells with
single cell form
(27)
. Therefore, increased density of
larger vessels may facilitate distant metastases by allowing the
intravasation and transportation of larger cancer cell clumps. In the
present study, patients with a high CDVI have a higher incidence of
distant metastasis after curative resection than patients with a low
CDVI (40% versus 0%, P = 0.005).
This in vivo observation in human is in line with that of
Liotta et al.(25, 26)
in mice.

The large intratumoral vessels collapse and dramatically decrease their
sizes from a living status to a fixed specimen
(28)
and
are seldom seen histologically, even when specifically sought
(29)
. Color Doppler ultrasound was reported to be able to
depict larger vessels of approximately 100 μm or larger in diameter
in vivo(23, 24)
. Therefore, the CDVI can then
provide better information on macrovessel density in living
state, which is not usually detectable in surgical-fixed specimens. As
a result, the CDVI can better reflect the tumor invasiveness,
recurrence, and prognosis.

Currently, MVD assessed with immunohistochemistry using antibodies
against various endothelial cell-related antigens such as factor
VIII, CD31, CD34 and so forth are widely used for assessing
angiogenesis in colorectal cancer
(10, 11, 30,
31,
32)
.
However, the results are inconsistent. Some studies
(10, 11, 30)
showed a significant association between the high MVD of
primary tumors and recurrence, metastasis, and survival. The study by
Bossi et al.(31)
and our present study didn’t
show any significant association. Surprisingly, Abdalla et
al.(32)
reported that high MVDs were associated with
a better prognosis in colorectal cancer. There seem to be some problems
to be resolved in MVD study. At first at laboratory level,
standardization of technique in MVD needs to be determined before
clinical application. Second, the inability of panendothelial
antibodies to distinguish between preexisting and newly formed blood
vessels could be an important factor in the assessment of true tumor
angiogenesis
(33, 34)
. Third, recent study has shown that
tumor cells themselves could substitute endothelial cells in tumor
tissue and panendothelial antibodies may not demonstrate this type of
neovascularization. Fourth, distribution of angiogenesis is usually
uneven and heterogenous, and the MVD represented microvessel counts in
tiny portions of tumor.

Colon cancer staging is still mostly performed according to the Dukes’
classification. However, the predictive value of tumor stage,
especially in the Dukes’ B and C categories (which are the stages most
patients belong to in this study), is rather limited
(35)
.
This may be the reason why the CDVI was significantly associated with
lymph node metastasis, positive vascular invasion, and poor prognosis
but was not correlated with Dukes’ stages in this study.

Vascular invasion is a well known factor for hematogenous metastasis
and also an independently prognostic factor in colorectal cancer
(36)
. Local shedding of cancer cells into the tumor
vascular stream that can commence at the onset of angiogenesis is
quantitatively related to the surface area of intratumoral vessels
(25, 37)
. The CDVI as a measure of global vascularity of
the tumor might reflect the surface area of intratumoral vessels. In
the present study, the patients with vascular invasion had a
significantly higher CDVI than those without. This may also explain why
hematogenous recurrence was more frequent in patients with a higher
CDVI and patients with a higher CDVI had poorer prognosis. Vascular
invasion is usually accompanied with lymphatic invasion that is
directly related to lymph node metastasis in colorectal cancer
(30, 36)
. In the present study, the CDVI, which was
correlated with vascular invasion, was also associated with lymph node
metastasis.

Various adjuvant therapies have been given to patients with advanced
colorectal cancer, including neoadjuvant and postoperative chemotherapy
or radiotherapy. The patients who need adjuvant therapies should be
selected by using some indicator affecting recurrence and prognosis.
The CDVI was shown to be an excellent prognostic indicator in colon
cancer patients. Thus, the CDVI in colon cancer patients obtained
before operation may help to identify patients with poor prognosis and
stratify patients for appropriate neoadjuvant therapy. Other than
chemotherapy or radiotherapy, TNP-470, an analogue of fumagillin
derived from Aspergillus jumigatus, has been shown to
inhibit angiogenesis and the growth of some tumors
(38,
39,
40)
. Such agents may be valuable in the adjuvant
therapy of colon cancer patients with high CDVI tumors.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.